CN102317452B - Farmesene synthase - Google Patents
Farmesene synthase Download PDFInfo
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- CN102317452B CN102317452B CN200980148985.XA CN200980148985A CN102317452B CN 102317452 B CN102317452 B CN 102317452B CN 200980148985 A CN200980148985 A CN 200980148985A CN 102317452 B CN102317452 B CN 102317452B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
Abstract
A new farnesene synthase was isolated from tomato. The farnesene synthase shows surprising properties with regard to the end products formed and its gene has, on a nucleotide level, low sequence identity with known farnesene synthase genes from other sources. The invention relates to isolated polynucleotides, polypeptides encoded by said polynucleotides, genetic constructs, vectors, hosts, in particular plants, harbouring such polynucleotides, polypeptides and genetic constructs, and seed derived from such plants.
Description
Summary of the invention
A kind of separation is newly from the Farmesene synthase of tomato.The dead end product that this Farmesene synthase is formed shows wonderful character, and its gene other Farmesene synthase gene order homogeny of originating with known on nucleotide level is low.The present invention relates to the polynucleotide of separation, by the polypeptide of described polynucleotide encoding, gene constructs, carries the carrier of this polynucleotide, polypeptide and gene constructs, host, particularly plant, and the seed that this kind of plant produces.
Summary of the invention
In first, provide the polynucleotide shown in SEQ ID NO:1 and its homologue.This nucleotide coding has Farmesene synthase activity, the polypeptide of specifically α-Farmesene synthase activity and/or β-Farmesene synthase activity.
In yet another aspect, the invention provides the polypeptide shown in SEQ ID NO:2 and its homologue.This polypeptide has Farmesene synthase activity, and specifically α-Farmesene synthase is active and/or β-Farmesene synthase is active.
In yet another aspect, the invention provides gene constructs, the carrier containing this gene constructs, and the host cell containing this gene constructs, wherein said gene constructs contains polynucleotide of the present invention.
In yet another aspect, the invention provides the transgenic plant containing this gene constructs or vegetable cell, and the seed of this kind of plant or vegetable cell generation.
In yet another aspect, provide the method preparing α-farnesene and/or β-farnesene, comprise and utilize polynucleotide of the present invention.
In also having one, provide a kind of method that in regulating plant, α-farnesene and/or β-farnesene produce, comprise the expression level of the polypeptide regulating polynucleotide encoding of the present invention.
See detailed description below and embodiment, the explanation in description taken together with the accompanying drawings and embodiment, will be easier to understand above-mentioned aspect of the present invention and other advantage.
Definition
In following description and embodiment, employ some terms.In order to provide this specification sheets and the clear of claims and consistent understanding, provide to give a definition, comprise these and defining the scope provided.Unless had definition in addition, all technology used herein and scientific terminology have usual the understood identical meanings of one skilled in the art of the present invention.The disclosure of all publications, patent application, patent and other documents all in this article complete quote for reference.
" Farmesene synthase " refers to the enzyme that bisphosphate method Buddhist nun ester can be transformed into α-farnesene and/or-farnesene.
" Farmesene synthase is active " refers to that Farmesene synthase catalysis bisphosphate method Buddhist nun ester of the present invention forms α-farnesene, β-farnesene or both enzymic activitys.Available enzyme activity test, such as, test determination described in embodiment is active.The amino acid sequence variation of Farmesene synthase of the present invention can have the biologic activity of required change, comprises such as, the reaction kinetics of change, substrate utilization degree, products distribution or other character, such as stereochemical nature.
" gene constructs " refers to polynucleotide molecule, normally double-stranded DNA, wherein can insert another polynucleotide molecule (property inserted polynucleotide molecule), such as but not limited to cDNA molecule.Gene constructs can transcribe this insertion polynucleotide molecule containing allowing, and optional this translation of transcript is become the necessary element of polypeptide.This property polynucleotide molecule inserted can derived from host cell, or can derived from different cell or biology, and/or can be recombination of polynucleotide.Once be positioned at host cell, this gene constructs can be integrated in host chromosome DNA.This gene constructs can be connected with carrier.
Term " gene " refers to containing becoming RNA molecule (such as mRNA) at Intracellular transcription, with the DNA sequence dna of suitable control region (such as promotor) operability connecting zone (transcriptional domain).Therefore gene can comprise the sequence that multiple operability connects, such as promotor, 5 ' leader sequence (it comprises the sequence such as participating in translation initiation), (protein) coding region (cDNA or genomic dna) and 3 ' non-translated sequence (comprising such as translational termination site).
" host cell " refers to protokaryon or eukaryotic cell.Typical prokaryotic host cell comprises colibacillary various bacterial strain.Typical eukaryotic host cell has vegetable cell, yeast cell, insect cell or zooblast.
" percent sequence identity " mean when two aminoacid sequences or two nucleotide sequences aligned time, occupy the amino acid of same corresponding position or the percentage ratio of Nucleotide.Want nucleic acid normally at least 120 Nucleotide of comparative sequences length, preferably 200 Nucleotide, the nucleic acid of preferred 300 Nucleotide composition.Want polypeptide normally at least 40 amino-acid residues of comparative sequences length, preferably 65 amino-acid residues, more preferably the polypeptide of 100 amino-acid residue compositions.Preferably arrange Comparison Method with Clustal W and measure " percent sequence identity ".Available CLC Bio, the software program " CLC Free Workbench3 " of Cambridge, MA, USA carries out vClustalW and arranges comparison.This program can from website
http:// www.clcbio.comon freely obtain.Nucleotide and amino acid range comparison parameter used are: Gap open penalizes 10 points, and breach extends penalizes 1 point.Nick End is identical with other breach all to be treated.
" polynucleotide " used herein refer to strand or the dichain polymer of deoxyribonucleotide or ribonucleotide bases, comprise DNA and corresponding RNA molecule, it can be sense or antisense orientation, consider to comprise cDNA, genomic dna and recombinant DNA, and the polynucleotide synthesized wholly or in part.Polynucleotide can be made up of whole gene or its any portion.Operability antisense polynucleotides can comprise the fragment of corresponding polynucleotide, and therefore the definition of " polynucleotide " comprises all this operability antisense fragments.
" polynucleotide of separation " are through identifying the nucleic acid molecule having polluted polynucleotide with its at least one previously combined and be separated.
" polypeptide " used herein comprises peptide, peptide and protein.
" isolated polypeptide " is through qualification and the polypeptide being substantially free of its natural surroundings component being separated or reclaiming.It comprises the polypeptides in situ in reconstitution cell.But, generally need by the polypeptide of at least one purification step preparative separation.
Term " genetic marker " or " polymorphism mark " or " molecule marker " refer to the region that genomic dna can be used for " mark " karyomit(e) specific position.If certain genetic marker and certain gene close linkage, or " in gene ", it " can mark " DNA containing this gene, thus can be used for molecule marker test (seeing below), selects or get rid of the existence of this gene in marker-assisted breeding/screening (MAS) method.The example of genetic marker has AFLP (fragment length polymorphism of amplification), micro-satellite, RFLP (restriction fragment length polymorphism), STS (sequencetagged site), SNP(Single nuclear polymorphism), SFP (single feature polymorphism; See Borevitz etc., 2003, genome research (Genome Research) 13:513-523), SCAR (Sequence Characterized amplification region), CAPS mark (the amplification polymorphism sequence of cutting) etc.Mark from gene more far away, the restructuring (exchange) between more possible occurrence flag and gene, thus lose chain (with mark with gene common-be separated).The distance measured between locus represents class frequency of attaching most importance to, and provides (cM, 1cM represents that between two kinds of marks, meiotic recombination frequency is 1%) with cM.Genomic size has a great difference between species, therefore being distributed in of recombination event whole genome is not random, and the physical distance (the kilobase kb namely between two kinds of marks) of in fact 1cM representative also has a great difference between species He in species.Should understand when digit synbol or " linked marker " herein, this also comprises the mark of this gene " interior " itself.
" molecule marker test " (or test) refers to the test of (based on DNA), can (directly or indirectly) to show in plant or a plant part whether there are polynucleotide of the present invention.
Detailed Description Of The Invention
An object of the present invention is to provide: the new polynucleotides sequence of coding Farmesene synthase, the enzyme of these nucleotide codings, external synthesis α-farnesene and/or-β farnesene, and/or genetically modified plant thus change the method for its α-farnesene and/or β-farnesene activity level, and provide the useful selection that can obtain enzyme to the public.
Be surprised to find by providing the separation polynucleotide shown in SEQ ID NO:1, or have at least 50% with nucleotide sequence shown in SEQ ID NO:1, preferred at least 60%, more preferably at least 70%, even more preferably 90%, the most preferably polynucleotide of at least 95% sequence thereto, and solve the problem.Preferred described polynucleotide encoding Farmesene synthase, namely can synthesize the enzyme of α-farnesene and/or β-farnesene from suitable substrates.Therefore, in another embodiment, provide polynucleotide of the present invention, wherein said polynucleotide encoding has the polypeptide of Farmesene synthase activity.
Nucleotide of the present invention shown in SEQ ID NO:1 is the encoding sequence of Farmesene synthase gene, the Farmesene synthase available from tomato plants money tree kind (Solanum lycopersicon cultivar Money Maker) of its coding as described in detail in method.As being hereafter described in further detail, Farmesene synthase of the present invention has wonderful character compared with Farmesene synthase known in the art, not only polynucleotide sequence homogeny is variant, and the activity of the importantly Farmesene synthase of this polynucleotide encoding is variant.
Those skilled in the art understood, and additionally provide and have at least 50% with nucleotide sequence shown in SEQ ID NO:1, and preferably at least 60%, more preferably at least 70%, even more preferably 90%, the most preferably polynucleotide of at least 95% sequence thereto.
In the context of the present invention, adopt ClustalW to arrange Comparison Method and measure sequence thereto.Adopt CLC Bio company, the software program " CLC Free Workbench3 " of Cambridge, MA, USA carries out ClustalW and arranges comparison.This program can from website
http:// www.clcbio.comon freely obtain.Nucleotide and amino acid range comparison parameter used are: Gap open penalizes 10 points, and breach extends penalizes 1 point.Nick End is identical with other breach all to be treated.
Described ClustalW is adopted to arrange Comparison Method, calculate polynucleotide shown in SEQ ID NO:1 and only have 36% homogeny with known being separated from the E-β-Farmesene synthase (Schee etc. 2002) of corn, have 46% homogeny with known being separated from the α-Farmesene synthase of Malus spectabilis housefly (Malus domesticus) (WO2004/035791), the E-β-Farmesene synthase of hybridizing pepper (Mentha x piperita) (WO99/18118) with known peppermint has 48% homogeny.
In another embodiment, the present invention also provides and in SEQ ID NO:1 polynucleotide sequence, contain 20,15,10,9,8,7,6,5,4,3,2 or 1 different polynucleotide inserting, lack or replace compared with polynucleotide shown in SEQ ID NO:1.These insert, lack or replacement can be contiguous nucleotides, also may reside in, such as, on 2,3,4 or 5 different positionss of SEQ ID NO:1 polynucleotide.Such as, can lack first Nucleotide, deputy two Nucleotide can be substituted, and insert one or more Nucleotide SEQ ID NO:1 polynucleotide the 3rd.
(separation) of the present invention polynucleotide can be cDNA forms, or its corresponding RNA, the polynucleotide of synthesis, also comprise the polynucleotide simultaneously containing intron and exon.Technician should be understood that polynucleotide of the present invention are except the region of code book invention Farmesene synthase, also can comprise regulating and controlling sequence, preferably include promoter region, the such as promoter region of Farmesene synthase of the present invention.
Additionally provide the fragment of polynucleotide of the present invention, as long as this fragment coding of polynucleotide of the present invention has the polypeptide of farnesene activity.It is active that available various method known in the art measures farnesene, but preferably measure farnesene activity by described in disclosed embodiment.
As mentioned above, found polynucleotide of the present invention, such as shown in SEQ ID NO:1, polynucleotide encoding has the polypeptide of farnesene activity.Specifically, found that polynucleotide encoding of the present invention can form the polypeptide of α-farnesene and/or β-farnesene.In this embodiment, adopt polynucleotide of the present invention to form α-farnesene and/or β-farnesene.
In addition, there is (+)-valencene (valencene) in result display, points out polypeptide of the present invention also to can be used for forming (+)-valencene.Therefore, in this article, when mentioning preparation polypeptide of the present invention, except α-Farmesene synthase activity and/or β-Farmesene synthase activity, it is active that polypeptide of the present invention also can show (+)-Valencene synthase, namely forms (+)-valencene.
α-farnesene can have four kinds of multi-form steric isomers: (Z, E)-α-farnesene, (Z, Z)-α-farnesene, (E, Z)-α-farnesene and (E, E)-α-farnesene.β-farnesene can have two kinds of multi-form steric isomers: (E)-β-farnesene and (Z)-β-farnesene.
In another embodiment, provide polynucleotide of the present invention, wherein this polynucleotide encoding has the polypeptide of α-Farmesene synthase activity and/or β-Farmesene synthase activity.
Be surprised to find polynucleotide of the present invention to encode polypeptide of the present invention, the latter can synthesize α-farnesene and/or β-both farnesenes, as shown in the Examples.On the contrary, other Farmesene synthase known to applicant, such as those enzymes disclosed in WO2004/035791and WO99/18118, be described to form α-farnesene or β-farnesene.Such as, polynucleotide optimized encoding of the present invention polypeptide of the present invention, the latter can synthesize (Z, E)-α-farnesene, (Z, Z)-α-farnesene, (E, Z)-α-farnesene and (E, E)-α-farnesene.
Such as, find that the polypeptide of polynucleotide encoding shown in SEQ ID NO:1 has synthesis α-farnesene and β-both farnesenes (specifically (E)-β-farnesene, (Z, E)-α-farnesene, (E, E)-α-farnesene, (E, Z)-α-farnesene and (Z, Z)-α-farnesene) activity.Now, Late Cambrian polynucleotide of the present invention, be such as separated the polynucleotide from tomato plants (money tree kind), the polypeptide of coding has the performance of synthesis α-farnesene steric isomer different from β-farnesene.In fact, previously do not report this tomato plants, as money tree kind, can synthesize α-farnesene and β-both farnesenes, be therefore wonderful.
Provide polynucleotide of the present invention first now, can be used for being formed α-farnesene or β-farnesene or both.In addition, the relation between these polynucleotide and its coded polypeptide is further illustrated with the polypeptide of this polynucleotide and coding first now, and the activity of its farnesene different from external synthesis in vivo.Such as, can understand, obtain that to have such as (raising) β-Farmesene synthase active, and the mutant not having (or reduction) α-Farmesene synthase activity is possible.The amino acid sequence variation of Farmesene synthase of the present invention also can have the biologic activity of required change, comprises such as, the reaction kinetics of change, substrate utilization degree, products distribution or further feature, such as stereochemical characteristics.
In addition, utilize polynucleotide of the present invention and polypeptide, now likely only with a kind of polynucleotide or polypeptide can in vivo with the specific mixture of external synthesis α-farnesene and/or β-farnesene.Such as, can by polynucleotide introduced plant of the present invention or bacterium, or in vitro in system with Peptide systhesis α-farnesene of the present invention and/or β-farnesene.
Utilize polynucleotide of the present invention, effectively can control the synthesis of different farnesene, as hereafter will more described in detail, due to the plant of available its gene transformation (such as) susceptible genotype, thus produce the transgenic plant that there is the synthesis of dissimilar farnesene and change.
In a preferred implementation of polynucleotide of the present invention, provide polypeptide shown in coding SEQ ID NO:2, or have at least 50% with the aminoacid sequence of SEQ ID NO:2, preferred at least 60%, more preferably at least 70%, even more preferably at least 90%, the polynucleotide of the most preferably separation of the polypeptide of at least 95% sequence thereto.
Specifically, find within the scope of the present invention, advantageously can utilize polypeptide shown in coding SEQ ID NO:2, or have at least 50% with the aminoacid sequence of SEQ ID NO:2, preferred at least 60%, more preferably at least 70%, even more preferably at least 90%, the polynucleotide of the most preferably separation of the polypeptide of at least 95% sequence thereto.
In another embodiment, the fragment of polynucleotide of the present invention is provided, this fragment coding has the polypeptide of α-Farmesene synthase activity and/or β-Farmesene synthase activity, and preferred described fragment coding has the polypeptide of α-Farmesene synthase activity and/or β-Farmesene synthase activity.
Without prejudice to scope of the present invention, should understand except above-mentioned polynucleotide, multiple modification can be carried out to polynucleotide of the present invention, and not make these modify the activity of polypeptide coded by the modified polynucleotide of material injury.Such as, as mentioned above, polynucleotide of the present invention containing modifying, as disappearance, can replace and inserting, and the activity of not polypeptide coded by substantial effect.Therefore, present invention also offers the fragment of the polynucleotide of the present invention of the polypeptide of coding display α-Farmesene synthase activity and/or β-Farmesene synthase activity (preferably).This fragment is not limited to the polynucleotide of the present invention of specific size or specific part, as long as have α-Farmesene synthase activity and/or β-Farmesene synthase activity (preferably).Technician is by adopting the method provided in embodiment, or other appropriate method known in the art, is not difficult to determine whether this fragment of polynucleotide of the present invention has required activity.
In another aspect of the present invention, relate to the polypeptide of polynucleotide encoding of the present invention.Specifically, provide polypeptide shown in coding SEQ ID NO:2, or arrange with ClustalW as mentioned above that comparison measures, at least 50% is had with SEQ ID NO:2 aminoacid sequence, preferred at least 60%, more preferably at least 70%, even more preferably at least 90%, the polynucleotide of the most preferably separation of the polypeptide of at least 95% sequence thereto.
In another embodiment, to present invention also offers compared with the polypeptide of SEQ ID NO:2 the not homopolypeptide containing 20,15,10,9,8,7,6,5,4,3,2 or 1 aminoacid insertion, disappearance or replacement in SEQ ID NO:2 peptide sequence.These insertions, disappearance or replacement can be the adjacent amino acids in SEQ ID NO:2 polypeptide, also may reside in the different positions in SEQ ID NO:2 polypeptide, such as 2,3,4 or 5 different positionss.Such as, the first amino acids in SEQ ID NO:2 polypeptide can lack, and a deputy amino acid can be substituted, and the 3rd can be inserted one or more Nucleotide.
In a preferred embodiment, polypeptide of the present invention has Farmesene synthase activity, and preferred α-Farmesene synthase is active and/or β-Farmesene synthase is active, and more preferably α-Farmesene synthase is active and β-Farmesene synthase is active.But this does not also mean that the those polypeptides of the present invention (such as mutant) that eliminating shortage these two kinds is active.
In another embodiment, α-farnesene and the β-Farmesene synthase polypeptide of separation is provided.The synthesis of this peptide species of the present invention to α-farnesene and β-both farnesenes all has activity, can measure by described in embodiment.As mentioned above, be separated first now and obtain this peptide species (from tomato plant (money tree kind)), and as described hereinly can utilize this polypeptide valuably.
In another embodiment, provide the fragment of polypeptide of the present invention, described fragment has α-Farmesene synthase activity and/or β-Farmesene synthase activity, and preferred α-Farmesene synthase is active and β-Farmesene synthase is active.
Without prejudice to scope of the present invention, should understand except aforementioned polypeptides, multiple modification can be carried out to polypeptide of the present invention, and not make these modify the activity of this polypeptide of material injury.Such as, as mentioned above, polypeptide of the present invention can contain this kind of modification, as aminoacid deletion, replacement and insertion, and the activity of not this polypeptide of substantial effect.Therefore, present invention also offers the fragment of polypeptide of the present invention, they have, and α-Farmesene synthase is active and/or (preferably and) β-Farmesene synthase is active.This fragment is not limited to the polypeptide of the present invention of specific size or specific part, as long as have, α-Farmesene synthase is active and/or β-Farmesene synthase is active.Technician, by adopting the method or other appropriate method known in the art that provide in embodiment, is not difficult to determine whether this fragment of the present invention has required activity.
Polypeptide of the present invention or its fragment, also can such as generation of for its antibody except its Farmesene synthase activity, and these antibody can be used for test example as the polypeptide of the present invention in plant.
In another aspect of the present invention, polynucleotide of the present invention can be utilized design and polynucleotide of the present invention, preferably with polynucleotide shown in SEQ ID NO:1, or the oligonucleotide of the DNA sequence dna of its part chain complementation, can mark used as hybridization probe, detect the homologous gene in screening-gene group DNA or cDNA library.Can under High stringency hybridisation condition with this probe hybridization, and coding gene product take part in Farmesene synthase activity, preferably there is the homologous sequence of α-Farmesene synthase and/or β-Farmesene synthase activity, comprise within the scope of the invention.
Generally speaking, high stringent condition refers to following (DNA) hybridization conditions: allow at least 50 Nucleotide, preferably the Nucleotide of about 200 or more and specific sequence are at about 65 DEG C, containing 1M salt of having an appointment, preferred 6xSSC or other contain in the solution of suitable ionic strength and hybridize, with with containing about 0.1M or following salt, preferred 0.2xSSC or other is containing the condition (Sambrook of solution washing with suitable ionic strength, Maniatis, Fritsch, molecular cloning, laboratory manual, CSHL Press, USA, 1987)).These conditions can detect the sequence with about 90% or higher sequence thereto.
In another aspect of the present invention, polynucleotide according to the present invention devise oligonucleotide, can used as the hybridization probe in DNA analysis.These probes can be used as molecule marker, with the plant genotype distinguished containing Farmesene synthase of the present invention and the plant genotype lacking this Farmesene synthase.This probe can be used as the auxiliary tools selected.
Such as, whether they can be used for detection exists in the method for the nucleotide sequence of the present invention of code book invention polypeptide in plant tissue or its nucleic acid samples, and the method comprises:
A. Plant tissue samples or nucleic acid samples is obtained,
B. whether there are the one or more marks be connected with polypeptide of the present invention by molecule marker analysis of experiments, wherein said mark test can detect sequence shown in SEQ ID NO:1, or have at least 50% with the nucleotide sequence of SEQ ID NO:1, preferred at least 60%, more preferably at least 70%, even more preferably at least 90%, the most preferably sequence of at least 95% sequence thereto.
In an optimal way of the present invention, according to polynucleotide design oligonucleotides of the present invention, used as amplified reaction, the such as primer of polymerase chain reaction (PCR), define amplified production and can show to there is Farmesene synthase in specified plant genotype, preferably there is Farmesene synthase of the present invention.
In a preferred embodiment, described primer is used for the amplification of selectivity restriction fragment, to identify that the AFLP be closely connected with Farmesene synthase of the present invention marks.Really, a kind of useful especially technology is AFLP technology, if Vos, P. etc. are at nucleic acids research (Nucleic Acids Research), and 1995, volume 23, No.21:
Now, in fact molecule marker can be produced from polynucleotide of the present invention.The nucleotide sequence provided can be used as the genetic marker of molecular breeding method and germplasm screening and/or specificity analysis really.This mark can be used for the mark helping the plant screening containing or do not contain polynucleotide of the present invention or polypeptide.
Therefore, provide at least 15 continuous nucleotides utilizing SEQ ID NO:1, or there is with it sequence of at least 90% nucleotide sequence identity, preferably as the SEQ ID NO:1 polynucleotide be separated, or have at least 50% with nucleotide sequence shown in SEQ ID NO:1, preferred at least 60%, more preferably at least 70%, even more preferably at least 90%, the most preferably molecule marker of the polynucleotide of at least 95% sequence thereto, or as polynucleotide described in any one of claim 7-10, preferably there is the molecule marker of the polynucleotide of α-Farmesene synthase activity and/or β-Farmesene synthase activity.
Additionally provide polynucleotide shown in SEQ ID NO:1, or have at least 50% with SEQ ID NO:1 nucleotide sequence, preferred at least 60%, more preferably at least 70%, even more preferably at least 90%, the most preferably purposes of the polynucleotide of at least 95% sequence thereto, this purposes is that the molecule (DNA) be connected with nucleotide sequence of the present invention marks for gene mapping display.
S known as technical staff, heredity (or molecule) mark is positioned at the gene or DNA sequence dna that on karyomit(e), known site is associated with specific gene or proterties.The variant produced due to sudden change observable in locus or change can be described it as.Genetic marker can be short dna sequence, and such as single base pair changes (Single nuclear polymorphism, SNP) sequence around, or long sequence, such as micro-satellite.
In the present invention, this mark can be polynucleotide shown in SEQ ID NO:1, or have 50% at least with SEQ ID NO:1 nucleotide sequence, preferred at least 60%, more preferably at least 70%, even more preferably at least 90%, the most preferably polynucleotide (fragment) of at least 95% sequence thereto.
Navigate in certain gene or near gene to genetic marker mapping; be not difficult to accomplish for general Protocols in Molecular Biology personnel; at Lefebvre; V. & A.M.Chevre. " instrument of labeled plant disease and insect pest gene: summary " .Agronomie15,1995 (1): 3-19; Michelmore, R.W. " handling the molecular method of Disease resistance gene ". plant pathology yearbook Annual Review of Phytopathology33 (1995): 393-427; Michelmore; R.W.; R.V.Kesseli & E.J.Ryder. " gene mapping of lettuce ". publish in: based on the mark (DNA-based markers in plants) of DNA in R.L.Phillips & I.K.Vasil (chief editor) plant; Kluwer Acad.Publishers; Dordrecht; 1994, pp. 223-239; Winter, P. & G.Kahl. " molecular marking technique of plant improvement ". microbiology and biotechnology world magazine (World Journal of Microbiology & Biotechnology), 1995,11 (4): 438-448.
The invention still further relates to the diagnostic kit that oligonucleotide of the present invention is housed, whether there is Farmesene synthase gene of the present invention for detecting in studied genotype.This diagnostic kit can be avoided adopting loaded down with trivial details test to screen the genotype forming or do not formed such as α-farnesene and/or β-farnesene.
The invention still further relates to containing polynucleotide sequence of the present invention, optimized encoding Farmesene synthase of the present invention, such as, there is the gene constructs of the polynucleotide sequence of the Farmesene synthase of α-Farmesene synthase activity and/or β-Farmesene synthase activity.This gene constructs preferably is contained in vegetable cell the regulating and controlling sequence with function, and described regulating and controlling sequence has: such as promotor, with polynucleotide, and the such as promotor of SEQ ID NO:1 polynucleotide encoding sequence homology of the present invention or allos.
In another embodiment, described gene constructs contains 5 '-3 of code book invention polypeptide ' the open reading frame polynucleotide of orientation.In another embodiment, described gene constructs contain can with the polynucleotide of code book invention polypeptide preferably hybridize under high stringency conditions 5 '-3 ' polynucleotide of orientation.
The invention still further relates to the DNA vector containing DNA construction of the present invention.Suitable carrier can be cloning vector, conversion carrier, and expression vector etc. are well known to those skilled in the art.
In addition, carry containing said gene construction, such as, containing SEQ ID NO:1 polynucleotide or its part, or the cell of the carrier of its homologue is within the scope of the present invention.And the cell carrying gene constructs of the present invention is within the scope of the present invention.
In an optimal way of the present invention, with standard transformation techniques polynucleotide of the present invention to be introduced in the genome of described plant and to obtain transgenic plant.Preferred described Expressed in Transgenic Plant has the Farmesene synthase of the present invention of function, such as, have the Farmesene synthase of aminoacid sequence shown in SEQ ID NO:2.
In yet another embodiment of the present invention, polynucleotide of the present invention are transferred to Heterologous System by the transformation technology of using well known, such as but not limited to melon, and tobacco, Arabidopis thaliana, potato, beet, Semen Brassicae campestris, cucumber, capsicum, eggplant, cotton, corn, pumpkin, in watermelon and lettuce.Also comprise the system such as bacterium and yeast.Polynucleotide of the present invention are introduced in this type systematic and can be produced such as polypeptide of the present invention, and in vivo or external utilize these polypeptide produce farnesene, preferred α-farnesene and β-farnesene.
In yet another embodiment of the present invention, provide the seed derived from transgenic plant of the present invention or vegetable cell, and such as containing the transgenic plant of polynucleotide of the present invention (such as SEQ ID NO:1) or the seed of vegetable cell.
In another aspect of the present invention, provide a kind of method preparing α-farnesene and/or β-farnesene, comprise the following steps: cultivate the cell having used polynucleotide genetic modification of the present invention, active with the α-Farmesene synthase and/or β-Farmesene synthase that provide enhancing; Bisphosphate method Buddhist nun is provided ester to cell with optional; And be separated the α-farnesene and/or β-farnesene that produce.The method also comprises cultivates derived from tomato, the particularly cell of tomato used in embodiment.
In another embodiment, provide a kind of method that regulating plant produces α-farnesene and/or β-farnesene, the method comprises raising or reduces the expression with the Farmesene synthase of peptide sequence of the present invention, and wherein said raising or reduction change the polynucleotide of coding said polypeptide expression by genetic modification realizes.
Polynucleotide as herein described and polypeptide and fragment thereof, and preferred coding has Farmesene synthase activity, those polynucleotide of preferred α-Farmesene synthase activity and/or β-Farmesene synthase activity and fragment thereof, there is multiple use, list in this article, but do not limit the scope of the invention.Such as, polynucleotide of the present invention can be used for producing the plant that (mistake) expresses Farmesene synthase of the present invention, give the improvement to aphid Behavior-Based control, and disperse herbivorous insect, such as aleyrodid.
Separation in an embodiment with regard to polynucleotide of the present invention is further described by the present invention.Lower embodiment just illustrates current the considered preferred forms of the present invention, and does not mean that restriction the present invention.
Technical field
The present invention relates to Farmesene synthase, specifically α-Farmesene synthase and/or β-Farmesene synthase.Also the polynucleotide sequence of encoding such enzymes is related to.The present invention also relates to the nucleic acid (or gene) construction, carrier and the host cell that are mixed with this polynucleotide sequence.Also relate to production of α-farnesene and/or β-farnesene and uses thereof.
Background technology
Term farnesene refers to that one group six kinds is all the closely-related compound of sesquiterpene.α-farnesene and β farnesene are isomer, have a position of double bond difference each other.
α-farnesene is 3,7,11-trimethylammonium-1,3,6,10-12 carbon tetraene, and β-farnesene is 7,11-dimethyl-3-methylene radical-1,6,10-12 carbon triolefin.There are four kinds of steric isomers in α-farnesene, difference is two the Geometry differences (steric isomer of the 3rd internal double bonds is identical) in three internal double bonds.There are two kinds of different steric isomers of central double bond Geometry in β-farnesene.Just be familiar with limited for the relation of the difference between α-farnesene and the various isomer of β-farnesene and these isomer concrete functions.
α-farnesene composing type is present in many species maybe can be induced (Gapper etc., results artifact and technology (Postharvest Biology and Technology) 42:(2006) 225-233).It is believed that α-farnesene is synthesized by hydro carbons intermediate reaction process by bisphosphate method Buddhist nun ester (FDP), and report is by the catalysis (Rupasinghe of α Farmesene synthase, Deng, J.Am.Soc.Hortic.Sci.123,882-886 (1998)).
β-farnesene is extensively present in various plant and animal.Published various about this natural product and in chemical communication is disposed as the document of important courier.β-farnesene is present in many gymnosperms and angiosperm, comprise german chamomile (Chamomilla recutita), grape (Vitis vinifera), corn (Zea mays), and in the essential oil of pepper (Piper nigrum).(E)-β-Farmesene synthase is that egyptian cotton floral leaf worm (Spodoptera littoralis) induces, this leaf worm colonizes in corn husk and leaf texture, but be not present in (Schnee etc. in root tissue, plant physiology (Plant Physiol), 2002,130:2049-2060).β-farnesene it is believed that it is by FDP by relating to the auxiliary bisphosphate ionization of divalent-metal ion, and synthesized by the carbocation C-3 methyl deprotonation reaction produced, see content disclosed in WO1999/18118.
The α described-farnesene function is as insect attractant, plays the effect of gender information's element in mouse and insect.It plays alarm pheromones effect (Sobotn í k etc., (2008) chemical ecology magazine J.Chem.Ecol., 34 (4): 478-86) in former reticulitermes lucifugus (Prorhinotermes canalifrons).Other purposes of α-farnesene and derivative thereof is strong cancer preventive, and for plastic film synthesis (US20060137032).Hern á ndez-ceruelos etc. (Toxicol.Lett.135:103-110,2005) report, camomile essence main body of oil β-farnesene has mutagenesis when mutagenic treatment bone marrow cells in mice).
It is reported that β-farnesene is the main component of lupine pollen smell, the pollination behaviour (Dobson etc., (1996) Am.J.Bot.83,877-885) of wasp can be stimulated.More the most important thing is, report aphid, such as cotton aphid (Aphis gossypii) can utilize β-farnesene as alarm pheromones (Jianwei etc., 2006, J.Econ.Entomol.99 (5): 1636-1640; Kislow and Edwards, 1972, natural Nature235:108-109; Pickett and Griffiths, 1980, J.Chem.Ecol.6:349-360), green black peach aphid worm (Myzus persicae) (Edwards, L.J., become uneasy after 1973, natural 241:126-127) contacting β-farnesene, and disperse (Wohlers (1981) Z Angew.Entomol.92,329-336) from the plant of its parasitism.β-farnesene has anxious toxicity ((1990) Acta Phytopathol.Entomol.Hung.25, the 331-342 such as van Oosten) when 100ng/ aphid dosage to aphid.Also interested is notice that β-farnesene steam is to aleyrodid toxic (Klijnstra etc., (1992) Meded Fac.Landbouwwet.57,485-491).Unfortunately, crops topical application β-farnesene is controlled to the effort seldom success of aphid behavior, because its volatile and rapid oxidation inactivation in atmosphere.(Dawson etc. (1988) Pest.Sci.22,17-30).
(2049 pages (2002) describe a kind of three diterpene synthases to Schnee etc. for plant physiology (Plant Physiology), 130 volumes, catalysis can form (E)-β-farnesene.WO2004/035791 discloses the α-Farmesene synthase of Malus spectabilis housefly (Malus domestica).WO99/18118 discloses (E)-β-Farmesene synthase of peppermint (Mentha piperita).
Embodiment
Vegetable material and trichome, RNA and mRNA is separated
Cultivate tomato plant (tomato money tree kind) in soil 4 weeks, day and night temperature is 23 DEG C-18 DEG C, and light/dark scheme is 16/8 hour.Cut same plant and contain merismatic stem, be placed in soil and cultivate 3 weeks again.After the stalk stem section of vortex concussion liquid nitrogen cryopreservation, stalk stem trichome is collected at the bottom of 50ml pipe.
Be separated the trichome RNA obtaining vegetable cell and tissue according to manufacturers protocol with the little test kit of Qiagen RNeasy plant (D ü sseldorf, Germany).Cracking is carried out with the RLT damping fluid provided.With multiple extracting (PolyAtract) the mRNA separation system III of Pu Luomaige (Madison, Wisconsin, USA) company according to manufacturers protocol, be separated from total serum IgE pond and obtain messenger RNA(mRNA).Being separated from total serum IgE the efficiency obtaining mRNA is 0.71%.
MRNA amplification and double-strand cDNA synthesis
With the MessageAmp II RNA amplification test kit amplification trichome mRNA of Ambion (Austin, Texas, USA) company.Input 100ng mRNA is used for amplification.Increase according to manufacturers protocol.The productive rate of cloning RNA is 171 μ g, and as expected, its scope is about 400-3.000 base to size distribution.
Often criticize in 20 μ l reactants containing 10 μ g RNA, with random hexamers (purchased from MWG Operon, Ebersberg, Germany) synthesize the first chain cDNA. from the RNA of amplification the RNA of the random primer of final concentration 62.5nM and amplification is merged, cultivate 5 minutes, be then stored on ice for 70 DEG C.Add the RevertAid M-MulV reversed transcriptive enzyme (200 unit) that mixing provides Fermentas Life Sciences (St.Leon-Rot, the Germany) company of damping fluid to some extent.Add the Nucleotide of final concentration 1mM, 42 DEG C are carried out cDNA and synthesize 90 minutes.Then, directly test tube is moved on on ice, carry out the synthesis of the second chain with Fermentas RNase H and e. coli dna polymerase I.DNA polymerase i reaction buffer and the RNaseH of 1 unit and the DNA polymerase i of 30 units that 8 μ l provide is added in each 20 μ l reaction tubess.With water, reaction volume is added to 100 μ l.Add all components of precooling, 15 DEG C are carried out the second chain and synthesize 2 hours.
Extensive parallel Manganic pyrophosphate complex initiation and data analysis
Carry out double-strand cDNA shearing with method known to the skilled described in this area, prepared by library, and 454 check order and adjoin arrangement comparison.Process relates to 454 order-checkings adapter (adapter) of bar code, 5 mutant strains are each carries a different bar code (see such as WO2007073165, WO2007037678 or WO2007073165, wherein different bar codes is called mark or marker).Manganic pyrophosphate complex initiation method itself is known in the art, at www.biotagebio.com; Also described by having in www.pyrosequencing.com/section technology.Such as at WO03/004690, WO03/054142, WO2004/069849, WO2004/070005, WO2004/070007, and in WO2005/003375 (all Dou Shi 454 Life Sciences Corp. have, and are incorporated herein for reference) also used this technology.The method that aligned sequences compares is well known in the art.The description of various program and alignment algorithm can be see: Smith and Waterman (1981) Adv.Appl.Math.2:482; Needleman and Wunsch (1970) J.MoI.Biol.48:443; Pearson and Lipman (1988) Proc.Natl.Acad.Sci.USA85:2444; Higgins and Sharp (1988) Gene73:237-244; Higgins and Sharp (1989) CABIOS5:151-153; Corpet et al. (1988) Nucl.Acids Res.16:10881-90; Huang etc., the computer utility 8:155-65 in (1992) bio-science; And Pearson etc., (1994) molecular biology method (Meth.MoI.Biol.) 24:307-31, is incorporated herein for reference.Altschul etc., (1994) natural genetics (Nature Genet.}6:119-29 (being incorporated herein for reference) considering in detail of providing that series arrangement comparison method and homology calculate.
The qualification of tomato sesquiterpene synthase SlSTS3 and clone
454-sequence reads the long 793bp of contig that (lcl-CL117Contig1) produces, and this contig and the stacked heath eggplant of squama (Fabiana imbricate) sesquiterpene synthase (not having known function) mRNA (AY860847) have the homogeny of higher degree.In order to obtain the full-length clone of this tomato cDNA, pcr amplification is carried out to the cDNA library built with tomato trichome mRNA.HybriZAP-2.1XR library construction Kit and the HybriZAP-2.1XR cDNA synthetic agent box of Stratagene (Cedar Creek, Texas, USA) company is adopted according to manufacturers protocol.The size of primary cDNA library is 2.5x10
6plaque forming unit (PFU)/μ g bacteriophage arm.To increase this primary library according to manufacturers protocol.With the cDNA library of the extensive cutting scheme cutting amplification described in manufacturer.PCR is made, 3 ' and 5 ' part of amplification cDNA fragment with the library after cutting and primer 1-6 (the primer table seen below in table 1).
Table 1 primer table
For the 5 ' part of this cDNA, carry out PCR with primer 6R5E and pActF (see primer table see primer list), PCR is carried out to 3 ' part primer 6F4E and T7 of this cDNA.With the cDNA library after cutting as template, the 0.25 unit Phusion hot start polysaccharase adopting Finnzymes (Espoo, Finland) company and the damping fluid provided, the concentration of each primer is 0.4mM, dNTP concentration is 0.2mM, and reaction volume is 25 μ l.MgCl is added in PCR mixture
2to final concentration 0.3mM.Employing Biometra (
germany) the T1 thermal cycler of company, and following program:
Step 1 98 DEG C 1 minute
Step 2 98 DEG C 10 seconds
Step 3 56 DEG C 30 seconds
Step 4 72 DEG C 45 seconds
Step 5 turns to step 2, repeats 34 times
Step 6 72 DEG C 5 minutes
Step 74 DEG C until take out from thermal cycler.
After dilute with water reaction product 20 times, be used as the template again increased.To increase again 5 ' part with primer 6R4E and pActF, by the long 3 ' part that increases again of primer 6F3E and T7.PCR condition is with PCR is identical first.
Cut again the PCR primer of amplified reaction from gel, be separated according to manufacturers protocol with the gel extraction agent box of QIAGEN company.With ABI Prism BigDye Terminator v1.1 cycle sequencing instrument, PCR primer is checked order.
By assembling overlapping sequence fragment, expand as complete encoding sequence by available from the contig of 454-sequence and the cDNA fragment of new order-checking.Devise new primer (primer E6flR2 and E6flF is shown in primer table) for PCR, the complete encoding sequence that the cDNA library after amplification cutting produces.Total length amplification is carried out as mentioned above with the Phusion hot start polysaccharase of Finnzymes company.
Cut current PCR from gel and react the PCR fragment obtained, be separated according to manufacturers protocol with the gel extraction agent box of QIAGEN.Then the TA Cloning Kit of Invitrogen (Carlsbad, California, USA) company is used the fragment of this purifying to be cloned in pCR2.1 cloning vector according to manufacturers protocol.Plasmid is transferred in One Shot TOP10 Competent Bacillus coli cells (providing with test kit).Start liquid culture, according to the GeneJET Plasmid Miniprep Kit separation quality grain of manufacturers protocol with Fermentas company, measure the sequence of inset in plasmid.Fig. 1 shows the position of this sequence and all primers.
With above-mentioned Phusion Taq polysaccharase, primer E6TopoF and E6TopoR increases the complete encoding sequence of this plasmid.PCR fragment in separating gel described above.Express test kit by described in manufacturers protocol with the directed TOP0 of the champion pET200 of Invitrogen company, this full length cDNA clone is entered in pET TOP0 carrier.The collection of illustrative plates of expression vector as shown in Figure 2.
Then, this plasmid is transferred in One Shot TOP10 Competent Bacillus coli cells (providing with test kit).Separation quality grain also checks order.Sequence and pCR2.1 cloning vector first (chain) full-length cDNA obtained that checks order is identical.The encoding sequence of SlSST3 is as shown in SEQ.ID.No:1.SEQ.ID.No:2 shows the aminoacid sequence of derivation.
At expression in escherichia coli SlSST3 albumen
PET200 Plastid transformation containing SlSST3 enters in BL21Star (DE3) One Shot Competent Bacillus coli cells.37 DEG C of incubated overnight liquid cultures.On next day, with 2.5ml overnight culture inoculation 50ml nutrient solution, and to be cultured to 600nm optical density(OD) be 0.5.Culture is transferred to room temperature, with ultimate density 1mM isopropyl ss-D-1-Thiogalactopyranoside (IPTG) abduction delivering of Roche (Basel, Switzerland) company.Adopt in pET200 carrier the culture containing false inset (R2R3 type MYB transcribes introduction (Genebank accession number AY705977), does not have terpene synthase activity) as negative control.Adopt the non-induced of these two kinds of cultures to contrast simultaneously.Induce after 3 hours, get two 1ml equal portions of each culture, centrifugal 15 minutes of 4 DEG C of 2000g, then inhale and abandon nutrient solution and collect residual cell.Use liquid nitrogen freezing cell precipitation, preserve until carry out enzyme test for-80 DEG C.
Measure the 600nm optical density(OD) of 1ml equal portions.Centrifugal 10 minutes of 4 DEG C of 2000g, inhale and abandon nutrient solution collecting cell.Managing a pipe induction equal portions cell of not inducing be resuspended in the terpene synthase damping fluid of 50 μ l/OD units (as Van Schie etc., molecular biology of plants Plant Mol Biol2007Jun with one; Described in 64 (3): 251-63) in.Cultivate 30 minutes and sonicated cells with 1 μ g N,O-Diacetylmuramidase on ice after, 4 DEG C of 12000g collect soluble protein fraction in centrifugal 30 minutes.All the other inductions are resuspended in 50 μ l/OD unit PBST (0.8%NaCl (w/v), 0.02%KCl (w/v), 0.144%Na with the cell precipitation of not inducing
2hPO
4(w/v), 0.02%KH
2pO
4(w/v) and 0.02% (v/v) Tween-20) in.All with in soluble protein fraction adding the 4X sample buffer (8%SDS (w/v) of 1/3 volume, 40% glycerine (w/v), 20% beta-mercaptoethanol (v/v), 240mM Tris-HCl pH6.8 and 0.08% tetrabromophenol sulfonphthalein (w/v)), sample is placed in boiling water 5 minutes.Get 15 μ l protein examples electrophoresis on 10% acrylamide gel.With the total protein on the induction of plasmid of Xylene Brilliant Cyanine G (0.25%CBB (w/v), 30% methyl alcohol (v/v) and 10% acetic acid) dyeing containing SlSST3 and myb transcription factor and the acrylamide gel of non-inducing cell.Fig. 3 shows the scintigram of this stained gel.
On 10% acrylamide gel, electrophoresis is containing the induction of SLSST3 plasmid and the total protein of non-inducing cell and soluble protein fraction.After sample electrophoresis, by all protein delivery traces on Nitrocellulose.Trace is closed 1 hour with the PBST solution containing 5% milk powder.Blot overnight is cultivated in the 10mlPBST solution of the five poly-His antibody (catalog number (Cat.No.) 34660, lot number 130167450) containing 5% milk powder and QIAGEN company.Wash trace three times with PBST, then containing 5% (w/v) milk powder and Pierce company, cultivate one hour in the PBST of the horseradish peroxidase goat anti-mouse IgG antibody of Rockford, IL, USA, then wash 3 times with PBST.Peroxidase activity is detected with enhanced chemiluminescence instrument (Amersham, Buckinghamshire, UK).Chemiluminescent photo as shown in Figure 4.
The functional analysis of SlSST3 albumen
Detect the sesquiterpene synthase activity in residue culture cell precipitation.By Van Schie etc., molecular biology of plants Plant Mol Biol.2007Jun; Sesquiterpene synthases test is carried out described in 64 (3): 251-63 (being incorporated herein for reference).
The protein of purifying with Ni-is by Van Schie etc., molecular biology of plants Plant Mol Biol.2007Jun; Described in 64 (3): 251-63 (being incorporated herein for reference), or by described in (2004) plant physiology Plant Physiol.135:2025-2037 (being incorporated herein for reference) such as Ament, analytical reaction product on the GG-MS TOF that DB-5 post is housed.
Fig. 5 shows the GC-MS mass spectrum of the ion 69 of SlSST3 and the Myb transcription factor protein of purifying with the Ni-that FPP measures, and wherein retention time rises to 436 seconds in 402 seconds.Adopt believable standard substance, reference ion spectrum and retention time, the product at peak 1,2,3 and 4 is accredited as (E) β-farnesene (peak 1), (Z, E) α-farnesene (peak 2), (E, E) α-farnesene and (Z, Z) α-farnesene (all in peak 3) and (E)-nerolidol (peak 4).
Fig. 7 shows with the further GC-MC color atlas of FPP Detection and Extraction from the protein of expression tomato Farmesene synthase Bacillus coli cells.Peak 1, beta-elemene (*).2. (E) β-farnesene.3. the sesquiterpene (*) of the unknown.4. (Z, E) α-farnesene.5. (+)-valencene (*).6. (E, E) α-farnesene.7. the sesquiterpene (*) of the unknown.(*). the peak that (*) indicates is according to Mass Spectrometric Identification, and other peaks all are according to the Identification of mass spectrum and pure standard substance retention time.
Pure (E) β-farnesene is taught friendship by Dr.W.Boland and is provided.Mass spectrum and the mass spectra peak 1 of this sesquiterpene are shown in shown in Fig. 6 a.The mixture of (Z, E) α-farnesene and (E, E) α-farnesene is available from Sigma company.The mass spectrum at (Z, E) α-farnesene and peak 2 is shown in shown in Fig. 6 b, and Fig. 6 c shows the mass spectrum at (E, E) α-farnesene and peak 3.(E) nerolidol is available from Sigma company.The mass spectrum at this sesquiterpene and peak 4 is shown in shown in Fig. 6 d.
These results show the SlSST3 coding farnesene synthetic enzyme of clone from tomato trichome cDNA, (ground of preponderating) has β-farnesene synthase activity, and extra α-farnesene is active.
The relative expression levels of tomato Farmesene synthase in various tissue and organ
In Fig. 8, measured by quantitative RT-PCR, show the space expression of (of the present invention) Farmesene synthase in tomato, and the cDNA that composing type RCE1 expresses at tomato different tissues is corrected.Bar graph represents two independent mean values judged.Error bars represents maximum value.Synthesized from: 1. piece, 2. petal, 3. stamen, 4. petal, 5. sepal, 6. prematurity Chinese olive, 7. broken fruit 8. turns to fruit, 9. haw, 10. trichome, 11. stems, 12. plants tops, 13. spires, 14. mature leafs, the cDNA of the RNA that 15. ageing leaves are separated.
Claims (14)
1. polynucleotide shown in the SEQ ID NO:1 be separated.
2. polynucleotide as claimed in claim 1, is characterized in that, described polynucleotide encoding has the polypeptide of α-Farmesene synthase activity and β-Farmesene synthase activity.
Isolated polypeptide shown in 3.SEQ ID NO:2.
4. polypeptide as claimed in claim 3, wherein said polypeptide has α-Farmesene synthase activity and β-Farmesene synthase activity.
5. the polynucleotide of the separation of coding polypeptide according to claim 3.
6. the gene constructs containing polynucleotide described in claim 1.
7. the gene constructs containing polynucleotide according to claim 5.
8. the carrier containing gene constructs described in claim 6.
9. the host cell containing gene constructs described in claim 6.
10. prepare a method for α-farnesene and/or β-farnesene, the method comprises the following steps: cultivate the cell having carried out genetic modification with polynucleotide described in claim 1, to provide the enhancing of α-Farmesene synthase and/or β-Farmesene synthase activity.
11. methods as claimed in claim 10, it is characterized in that, the method also comprises: provide bisphosphate method Buddhist nun ester to cell; And be separated the α-farnesene and/or β-farnesene that produce.
12. 1 kinds of regulating plants produce the method for α-farnesene and/or β-farnesene, the method comprises raising or reduces the expression with the Farmesene synthase of peptide sequence described in claim 4, and wherein said raising or reduction change the expression of the polynucleotide of coding said polypeptide by genetic modification and realize.
13. 1 kinds are detected in plant tissue or its nucleic acid samples the method that whether there is the nucleotide sequence of polypeptide described in claim 3 of encoding, comprising:
A. Plant tissue samples or nucleic acid samples is obtained,
Whether b. exist with sample described in molecule marker analysis of experiments and mark with one or more of polypeptide associated described in claim 3, wherein said mark test detects sequence shown in SEQ ID NO:1.
Polynucleotide purposes shown in 14.SEQ ID NO:1 is for gene mapping, shows the DNA molecular marker be connected with SEQ ID NO:1 nucleotide sequence.
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| CA2305629A1 (en) | 1997-10-06 | 1999-04-15 | Washington State University Research Foundation | Isolation and expression of farnesene synthase from peppermint, mentha x piperita, l. |
| US20050009022A1 (en) | 2001-07-06 | 2005-01-13 | Weiner Michael P. | Method for isolation of independent, parallel chemical micro-reactions using a porous filter |
| US6902921B2 (en) | 2001-10-30 | 2005-06-07 | 454 Corporation | Sulfurylase-luciferase fusion proteins and thermostable sulfurylase |
| NZ523384A (en) * | 2002-12-24 | 2005-12-23 | Horticulture & Food Res Inst | Enzymes and polynucleotides encoding the same |
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| FR2914652B1 (en) * | 2007-04-03 | 2013-01-18 | Librophyt | GENES ENCODING Z, Z-FARNESYL DIPHOSPHATE SYNTHASE AND MULTI-PRODUCT SESQUITERPENE SYNTHASE AND USES THEREOF |
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- 2009-12-02 EP EP09771418.2A patent/EP2373790B1/en not_active Not-in-force
- 2009-12-02 CA CA2745853A patent/CA2745853A1/en not_active Abandoned
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004035791A1 (en) * | 2002-10-15 | 2004-04-29 | The Horticulture And Food Research Institute Of New Zealand Limited | Plant alpha farnesene synthase and polynucleotides encoding same |
Non-Patent Citations (3)
| Title |
|---|
| Evidence for Differential Folding of Farnesyl Pyrophosphate in the Active Site of Aristolochene Synthase: A Single-Point Mutation Converts Aristolochene Synthase into an (E)-β-Farnesene Synthase;Athina Deligeorgopoulou et al.;《Biochemistry》;20031231;第42卷(第25期);全文 * |
| Molecular scaffolds for chemical wizardry: Learning nature’s rules for terpene cyclases;Bryan Greenhagen et al.;《PNAS》;20011120;第98卷(第24期);全文 * |
| The Maize Gene terpene synthase 1 Encodes a Sesquiterpene Synthase Catalyzing the Formation of (E)-β-Farnesene,(E)-Nerolidol,and (E,E)-Farnesol after Herbivore Damage;Christiane Schnee et al.;《Plant Physiology》;20021231;第130卷;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| IL213393A0 (en) | 2011-07-31 |
| EP2373790A1 (en) | 2011-10-12 |
| AU2009323128B2 (en) | 2015-01-22 |
| AU2009323128A1 (en) | 2010-06-10 |
| CN104611360B (en) | 2017-06-30 |
| ES2535722T3 (en) | 2015-05-14 |
| CA2745853A1 (en) | 2010-06-10 |
| MY159141A (en) | 2016-12-15 |
| WO2010064897A1 (en) | 2010-06-10 |
| JP2012510811A (en) | 2012-05-17 |
| MX2011005872A (en) | 2011-08-17 |
| IL213393A (en) | 2017-10-31 |
| RU2011127438A (en) | 2013-01-10 |
| CN104611360A (en) | 2015-05-13 |
| US8674175B2 (en) | 2014-03-18 |
| KR20110097924A (en) | 2011-08-31 |
| WO2010064897A8 (en) | 2010-09-02 |
| JP5777523B2 (en) | 2015-09-09 |
| KR101696173B1 (en) | 2017-01-13 |
| RU2546877C2 (en) | 2015-04-10 |
| EP2373790B1 (en) | 2015-02-11 |
| CN102317452A (en) | 2012-01-11 |
| US20110307972A1 (en) | 2011-12-15 |
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